通过 CRISPR-Cas9 在簇特异性连接子相关序列上的基因编辑策略,探索 KRAB-Zfp 簇与其目标转座元件之间的调控关系。

IF 4.7 2区 生物学 Q1 GENETICS & HEREDITY
Yang Zhang, Fei He, Yanning Zhang, Qian Dai, Qintong Li, Jing Nan, Ruidong Miao, Bo Cheng
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引用次数: 0

摘要

背景:Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs)是哺乳动物中最大的超家族转录因子,主要靶向和抑制转座元件(TE)。由于这些转录调节因子的序列具有相似性和多样性,而且它们的靶向TE序列具有复杂的重复性,因此要剖析它们的不同功能具有挑战性:结果:小鼠KRAB-Zfps在全基因组范围内主要分为几个簇。结果:小鼠 KRAB-Zfps 主要在全基因组范围内组成一个簇,该研究发现簇内成员具有密切的进化关系,并具有类似的锌指(ZnF)使用偏好。KRAB-Zfps以细胞类型或组织类型特异性的方式表达,它们往往与其他簇成员一起被积极转录。进一步的序列分析表明,锌指之间的连接序列是保守的,同时具有独特的簇特异性。根据 KRAB-Zfp 簇的这些独特性,我们设计了 sgRNAs 来编辑簇特异性连接子,以取消目标簇的功能。我们以小鼠胚胎干细胞(mESC)为模型,筛选并获得了一系列靶向各种高表达 KRAB-Zfp 簇的 sgRNAs。我们在专为多靶点 sgRNAs 开发的报告分析法中验证了 sgRNAs 的有效性,并通过基于 PCR 的分析进一步证实了其有效性。利用诱导表达 Cas9 和这些 sgRNA 的 mESC 细胞系,我们发现编辑不同的 KRAB-Zfp 簇会导致不同类别 TE 的转录变化:总之,本研究发现的簇内 KRAB-Zfp 成员的内在序列相关性表明,在簇的进化过程中,保守的簇特异性连接子在串联 ZnF 阵列的多样化和 KRAB-Zfps 的相关靶特异性方面发挥了关键作用。在此基础上,研究人员开发并验证了一种基于CRISPR-Cas9的有效方法,该方法可针对连接子序列快速编辑KRAB-Zfp簇,从而确定簇成员与其潜在TE靶标之间的调控相关性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.

Exploration of the regulatory relationship between KRAB-Zfp clusters and their target transposable elements via a gene editing strategy at the cluster specific linker-associated sequences by CRISPR-Cas9.

Background: Krüppel Associated Box-containing Zinc Finger Proteins (KRAB-ZFPs), representing the largest superfamily of transcription factors in mammals, are predicted to primarily target and repress transposable elements (TEs). It is challenging to dissect the distinct functions of these transcription regulators due to their sequence similarity and diversity, and also the complicated repetitiveness of their targeting TE sequences.

Results: Mouse KRAB-Zfps are mainly organized into clusters genomewide. In this study, we revealed that the intra-cluster members had a close evolutionary relationship, and a similar preference for zinc finger (ZnF) usage. KRAB-Zfps were expressed in a cell type- or tissue type specific manner and they tended to be actively transcribed together with other cluster members. Further sequence analyses pointed out the linker sequences in between ZnFs were conserved, and meanwhile had distinct cluster specificity. Based on these unique characteristics of KRAB-Zfp clusters, sgRNAs were designed to edit cluster-specific linkers to abolish the functions of the targeted cluster(s). Using mouse embryonic stem cells (mESC) as a model, we screened and obtained a series of sgRNAs targeting various highly expressed KRAB-Zfp clusters. The effectiveness of sgRNAs were verified in a reporter assay exclusively developed for multi-target sgRNAs and further confirmed by PCR-based analyses. Using mESC cell lines inducibly expressing Cas9 and these sgRNAs, we found that editing different KRAB-Zfp clusters resulted in the transcriptional changes of distinct categories of TEs.

Conclusions: Collectively, the intrinsic sequence correlations of intra-cluster KRAB-Zfp members discovered in this study suggest that the conserved cluster specific linkers played crucial roles in diversifying the tandem ZnF array and the related target specificity of KRAB-Zfps during clusters' evolution. On this basis, an effective CRISPR-Cas9 based approach against the linker sequences is developed and verified for rapidly editing KRAB-Zfp clusters to identify the regulatory correlation between the cluster members and their potential TE targets.

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来源期刊
Mobile DNA
Mobile DNA GENETICS & HEREDITY-
CiteScore
8.20
自引率
6.10%
发文量
26
审稿时长
11 weeks
期刊介绍: Mobile DNA is an online, peer-reviewed, open access journal that publishes articles providing novel insights into DNA rearrangements in all organisms, ranging from transposition and other types of recombination mechanisms to patterns and processes of mobile element and host genome evolution. In addition, the journal will consider articles on the utility of mobile genetic elements in biotechnological methods and protocols.
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